Cellular analysis of body fluids

ABSTRACT

Herein is provided a simple, reliable and accurate method for cellular analysis on hematology analyzers. In various aspects, the methods provide separation and/or differentiation between red blood cells (RBCs) and white blood cells (WBCs) by utilizing a fluorescent dye to selectively stain WBCs such that they emit stronger fluorescence signals. The method provides optimal detection limits on WBCs and RBCs, thereby allowing analysis of samples with sparse cellular concentrations. As few as one reagent may be used to prepare a single dilution for body fluid analysis, in order to simplify the body fluid analysis. Minimal damage to WBCs is attained using the lysis-free approach described in aspects of the disclosure.

CROSS REFERENCE TO RELATED APPLICATIONS

Under 35 U.S.C. § 119(e), this application claims priority benefit tothe filing date of U.S. Provisional Patent Application Ser. No.61/580,623, filed on Dec. 27, 2011, the disclosure of which applicationis herein incorporated by reference in its entirety.

BACKGROUND

A variety of methods are used for cellular analysis, including visualand/or automated inspection via light or fluorescent light microscopy.Cellular examinations and analyses of these types are commonly practicedin order to obtain information regarding cell lineage, maturationalstage, and cell counts in a sample.

Flow cytometry is a method for identifying and distinguishing betweendifferent cell types in a non-homogeneous sample. In the flow cytometer,cells are passed one at a time or nearly one at a time through a sensingregion where each cell is irradiated by an energy source. Typically,single wavelength light sources (e.g., lasers, etc.) are used as theenergy source and one or more of a variety of sensors record data basedon the interaction of the cells with the applied energy. Flow cytometryis commonly used in hematology and has been particularly successful inthe diagnosis of blood cancers. In addition to flow cytometry, otheranalytical methods are used in hematology and in characterizing apopulation of cells.

Blood samples tend to have a high concentration of cells. Analysis ofsamples with significantly lower concentrations of cells, whether byflow cytometry or other techniques, is more difficult and therefore lesscommon. In addition, traditional hematology analyzers, which aredesigned to measure whole blood samples, tend to have limited detectionsensitivity for low-end cell concentrations. In some cases, manualexamination of samples is the only available method for cellularanalysis. Improved methods for analyzing samples with low cell countsare desirable in the fields of medicine, microbiology, and others.

SUMMARY

In one aspect, the disclosure provides a method for analyzing a bodyfluid containing cells, the method comprising: staining the body fluidwith a fluorescent dye, wherein the fluorescent dye permeates a cellmembrane and binds to a nucleic acid to form a dye complex within thecell; irradiating the stained body fluid with energy from an energysource; and measuring a fluorescence signal emitted by the dye complexin the stained body fluid.

In some such aspects, the body fluid comprises less than about 20cells/μL.

In some such aspects, the body fluid comprises more than about 20cells/μL.

In some such aspects, the nucleic acid is selected from a DNA and RNA.

In some such aspects, the energy source provides monochromatic lighthaving a wavelength in the visible spectrum, and wherein the wavelengthof the monochromatic light and the wavelength of the fluorescence signalare different.

In some such aspects, unbound fluorescent dye emits less fluorescentlight when irradiated with energy from the energy source compared withthe dye complex.

In some such aspects, unbound fluorescent dye does not fluoresce whenirradiated with energy from the energy source while unbound to thenucleic acid, such that cells lacking the dye complex do not emit afluorescence signal.

In some such aspects, the method comprises differentiating cells withnuclei from cells without nuclei based on the presence or absence of thefluorescent dye.

In some such aspects, the measuring involves enumerating anddifferentiating RBCs and WBCs.

In some such aspects, the method does not involve lysing RBCs prior tothe measuring.

In some such aspects, the body fluid comprises intact WBCs and RBCs.

In some such aspects, the measuring is carried out using an automatedhematology analyzer, flow cytometer, or other diagnostic analyzer forbody fluid samples.

In some such aspects, the measuring comprises flowing the body fluidthrough a flow cell in a cytometer.

In some such aspects, the fluorescent dye is provided in a compositionthat further comprises water.

In another aspect, the disclosure provides a method for analyzing afluid, wherein the fluid contains less than about 40 cells/μL, themethod comprising: contacting the fluid with a fluorescent dye, whereinthe fluorescent dye permeates a cell membrane and binds to a nucleicacid to form a dye complex within the cell; irradiating the fluid withenergy from an energy source; and measuring a fluorescence signalemitted by the dye complex in the fluid.

In some such aspects, the fluid contains less than about 20 cells/μL.

In some such aspects, the fluid contains less than about 5 cells/μL.

In some such aspects, the fluid is a body fluid.

In some such aspects, the fluid is a biological fluid.

In another aspect, the disclosure provides a method for differentiatingcells, the method comprising: contacting the cells with a solutioncomprising a fluorescent dye, wherein the fluorescent dye is watersoluble, capable of permeating a cell membrane, and capable of bindingto a nucleic acid; irradiating the cells with an excitation light froman excitation light source; and measuring light emissions from the cellsand differentiating cells containing nucleic acids from cells lackingnucleic acids based on the measuring.

In another aspect, the disclosure provides a composition for analyzing abody fluid, the composition comprising water and a fluorescent dye,wherein the fluorescent dye is water soluble, capable of permeating acell membrane, and capable of binding to a nucleic acid.

In another aspect, the disclosure provides a hematology systemcomprising: a sample holder for holding a sample to be analyzed; amixing receptacle for mixing at least a portion of the sample to beanalyzed with a staining composition; a storage receptacle for storingthe staining composition, wherein the staining composition comprises adye capable of permeating a cell membrane and binding to a nucleic acid,and wherein the dye is fluorescent when bound to a nucleic acid; a flowcell; at least one energy source for applying electromagnetic energy tothe flow cell; one or more detectors for detecting fluorescenceoriginating from within the flow cell; and one or more visible lightdetectors for detecting scattered visible light.

In some such aspects, the system comprises an aspirating mechanism foraspirating at least a portion of the sample to be analyzed from thesample holder to the mixing receptacle.

In some such aspects, the system comprises an aspirating mechanism foraspirating staining composition from the storage receptacle to themixing receptacle.

In some such aspects, the at least one energy source providesmonochromatic light at a wavelength λ1.

In some such aspects, the dye absorbs light at wavelength λ1 and emitslight at wavelength λ2 when the dye is bound to a nucleic acid.

In some such aspects, the detector detects light at wavelength λ2.

In some such aspects, the system comprises at least one additionalenergy source for providing non-monochromatic visible light.

In some such aspects, the sample to be analyzed is a body fluid.

In another aspect, the disclosure provides a method for preparing ahematology system, the method comprising: providing a mixing receptaclefor mixing at least a portion of a sample to be analyzed with a stainingcomposition; providing a storage receptacle for storing the stainingcomposition, wherein the staining composition comprises a dye capable ofpermeating a cell membrane and binding to a nucleic acid, and whereinthe dye is fluorescent when bound to a nucleic acid; providing a flowcell; positioning an energy source for providing electromagnetic energyto the flow cell; positioning a detector for detecting fluorescenceemitted from within the flow cell.

In another aspect, the disclosure provides a method for preparing areceptacle for use in analyzing a body fluid, the method comprisingadding to the receptacle a composition comprising a dye capable ofpermeating a cell membrane and binding to a nucleic acid, wherein thedye is fluorescent when bound to a nucleic acid, and wherein thereceptacle is an integrated or modular part of a hematology system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic image of an aspect of a body fluid analysismethod described herein. WBCs are separated from RBCs upon DNA-dyeinteraction and subsequent emission of fluorescence. The fluorescenceinformation, as well as other optical scattering signals, are used incellular analysis of body fluid samples. FL1 indicates the fluorescencecollected with a 530±20 nm band path filter. ALL and IAS are the lightscattering signals collected at 0 to 1 degrees and 3 to 10 degrees,respectively.

FIG. 2A is a scattergram (ALL v. IAS) of cellular analysis for a bodyfluid sample. WBC=89/μL (reference=81/μL);RBCs=2012/μL(reference=1733/μL).

FIG. 2B is a scattergram (FL1 v. ALL) of cellular analysis for a bodyfluid sample. WBCs and RBCs are well separated in fluorescence (FL1).WBC=89/μL (reference=81/μL); RBC=2012/μL (reference=1733/μL).

FIG. 3A is a scattergram (ALL v. IAS) of cellular analysis of anotherbody fluid sample. WBC=0.95/μL (reference=0.33/μL); RBC=142/μL(reference=122/μL).

FIG. 3B is a scattergram (FL1 v. ALL) of cellular analysis of anotherbody fluid sample. WBCs and RBCs are well separated in fluorescence(FL1). WBC=0.95/μL (reference=0.33/μL); RBC=142/μL (reference=122/μL).

FIG. 4A is an FL1 v. IAS scattergram of the first dilution of a sixlevel serial dilution of a buffy coat sample. The original buffy coatsample contains 6.1×10³/μL WBC and 15×10³/μL RBC. The six levels, (A),(B), (C), (D), (E) and (F), were prepared based upon 1:10, 1:30, 1:100,1:300, 1:1000 and 1:3000, respectively, dilutions with PBS. All sampleswere measured using a dilution on a prototype analyzer.

FIG. 4B is an FL1 v. IAS scattergram of the second dilution of a sixlevel serial dilution of a buffy coat sample. The original buffy coatsample contains 6.1×10³/μL WBC and 15×10³/μL RBC. The six levels, (A),(B), (C), (D), (E) and (F), were prepared based upon 1:10, 1:30, 1:100,1:300, 1:1000 and 1:3000, respectively, dilutions with PBS. All sampleswere measured using a dilution on a prototype analyzer.

FIG. 4C is an FL1 v. IAS scattergram of the third dilution of a sixlevel serial dilution of a buffy coat sample. The original buffy coatsample contains 6.1×10³/μL WBC and 15×10³/μL RBC. The six levels, (A),(B), (C), (D), (E) and (F), were prepared based upon 1:10, 1:30, 1:100,1:300, 1:1000 and 1:3000, respectively, dilutions with PBS. All sampleswere measured using a dilution on a prototype analyzer.

FIG. 4D is an FL1 v. IAS scattergram of the fourth dilution of a sixlevel serial dilution of a buffy coat sample. The original buffy coatsample contains 6.1×10³/μL WBC and 15×10³/μL RBC. The six levels, (A),(B), (C), (D), (E) and (F), were prepared based upon 1:10, 1:30, 1:100,1:300, 1:1000 and 1:3000, respectively, dilutions with PBS. All sampleswere measured using a dilution on a prototype analyzer.

FIG. 4E is an FL1 v. IAS scattergram of the fifth dilution of a sixlevel serial dilution of a buffy coat sample. The original buffy coatsample contains 6.1×10³/μL WBC and 15×10³/μL RBC. The six levels, (A),(B), (C), (D), (E) and (F), were prepared based upon 1:10, 1:30, 1:100,1:300, 1:1000 and 1:3000, respectively, dilutions with PBS. All sampleswere measured using a dilution on a prototype analyzer.

FIG. 4F is an FL1 v. IAS scattergram of the sixth dilution of a sixlevel serial dilution of a buffy coat sample. The original buffy coatsample contains 6.1×10³/μL WBC and 15×10³/μL RBC. The six levels, (A),(B), (C), (D), (E) and (F), were prepared based upon 1:10, 1:30, 1:100,1:300, 1:1000 and 1:3000, respectively, dilutions with PBS. All sampleswere measured using a dilution on a prototype analyzer.

FIG. 5 provides correlation graphs of WBC (measured vs. calculated)among all six levels of diluted buffy coat samples (A) and three low-endsamples only (B). Multiple dots at each level indicate that multipleruns were performed. The overall correlations were: Y=1.0239X−4.9(R²=0.9984) for all six levels and Y=1.0787X−1.5 (R²=0.9598) for thethree levels with the lowest cell concentrations.

FIG. 6 provides correlation graphs of WBC (current method vs. reference)for 91 body fluid samples. The correlations were plotted in differentranges: (A) full range (˜40,000/μL), (B)<2,000/μL, (C) <200/μL and (D)<50/μL. The dilution ratio was 1:35 (specimen to labeling reagent).

FIG. 7 provides correlation graphs of RBC (current method vs. reference)for 91 body fluid samples. The correlations were plotted in differentranges: (A) full range (˜200,000/μL), (B) <3,000/μL, (C) <200/μL and (D)<50/μL. Samples with many RBC ghosts were not included in thecorrelation analysis. The dilution ratio was 1:35 (specimen to labelingreagent).

FIG. 8 is a correlation graph of WBC (invented method vs. reference) for72 body fluid samples with very low cell concentrations (WBC<50/μL). Thedilution ratio was 1:10 (specimen to labeling reagent).

FIG. 9 is a correlation graph of RBC (current method vs. reference) for38 body fluid samples with very low cell concentrations (RBC<50/μL).Samples with many RBC ghosts were not included in correlation analysis.The dilution ratio was 1:10 (specimen to labeling reagent).

DETAILED DISCLOSURE

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are described herein. It isunderstood that the present disclosure supersedes any disclosure of anincorporated publication to the extent there is a contradiction.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The term “typically” is used to indicate common practices of theinvention. The term indicates that such disclosure is exemplary,although (unless otherwise indicated) not necessary, for the materialsand methods of the invention. Thus, the term “typically” should beinterpreted as “typically, although not necessarily.” Similarly, theterm “optionally,” as in a material or component that is optionallypresent, indicates that the invention includes instances wherein thematerial or component is present, and also includes instances whereinthe material or component is not present.

As used herein, the term “body fluid” refers to fluids present orobtained from an animal, including fluids such as cerebrospinal fluid,peritoneal fluid, pericardial fluid, pleural fluid, synovial fluid,urine, saliva, tears, semen, amniotic fluid, sputum, and the like, aswell as fluids obtained from cysts, tumors, and the like. Unlessotherwise specified, the term “body fluid” does not include whole blood,although a body fluid may contain red blood cells (RBCs) and/or whiteblood cells (WBCs).

In some aspects, the disclosure provides methods and materials foranalyzing fluids with low cell count. By low cell count is meant fluidsthat have less than about 100 cells/μL, or less than about 80 cells/μL,or less than about 60 cells/μL, or less than about 40 cells/μL, or lessthan about 30 cells/μL, or less than about 20 cells/μL, or less thanabout 10 cells/μL, or less than about 5 cells/μL. In some embodiments,such low cell count is the result of dilution of a more concentratedoriginal sample. In other embodiments, such low cell count is a naturalcharacteristic of the fluid to be analyzed (i.e., no dilution isnecessary to achieve the low cell count).

The fluids to be analyzed using the methods and materials describedherein are, in some embodiments, selected from a body fluid. In otherembodiments, the fluids to be analyzed are biological in nature but arenot body fluids. In some embodiments, the fluid to be analyzed is“synthesized,” meaning that a population of cells is added to a fluid,wherein the fluid is biologically compatible but is not the fluid inwhich the cells are normally found. Examples of such fluids includebuffered aqueous solutions and the like.

Materials

In some embodiments, the methods of interest involve contacting apopulation of cells with a labeling composition suitable to aid in thedesired characterization of the cells. In some embodiments the labelingcomposition comprises a fluorescent dye, and optionally furthercomprises additional components such as those described herein below.The components of the labeling composition and the relativeconcentration of such components may be varied according to the needsand requirements of the intended use. The labeling composition, with orwithout the dye present, may alternatively be referred to herein as“reagent.”

The methods and materials of interest involve a fluorescent dye (alsoreferred to herein as a “dye”). The fluorescent dye may be any suitabledye having the characteristics necessary or suitable to carry out themethods of interest. For example, in some embodiments, the dye is watersoluble. For example, in some embodiments, the fluorescent dye has anaqueous solubility of at least 1 μg/L, or at least 5 μg/L, or at least10 μg/L, or at least 20 μg/L, or at least 50 μg/L. In some embodiments,the dye is stable in aqueous solutions, meaning that the dye does notsignificantly degrade on the timescale suitable for the methods ofanalysis described herein. For example, the dye is stable in a bufferedaqueous solution (e.g., cell reagent) at ambient temperatures (e.g.,approximately 20-25° C.) for at least 30 min, or at least 1 hr or atleast 6 hr, or at least 12 hr, or at least 24 hours, or at least 2 days,or at least 7 days, or at least 1 month. Also for example, the dye isstable in a buffered aqueous solution (e.g., cell reagent) underrefrigeration (e.g., below approximately 10° C.) for at least 1 hr, orat least 12 hr, or at least 24 hours, or at least 2 days, or at least 7days, or at least 1 month, or at least 6 months, or at least 12 months.

In some embodiments, the dye is able to penetrate a cell membrane, suchas the walls of blood cells or the like. In some embodiments, the dye isfurther able to penetrate a cell nucleus once inside a cell.

In some embodiments, the dye binds to a nucleic acid. In someembodiments, the dye binds to DNA. In some embodiments, the dyes ofinterest bind to DNA but do not bind to RNA. In some embodiments, thedyes of interest bind preferentially to DNA over RNA. In someembodiments, binding of the dye to a DNA molecule involveshydrogen-bonding or another non-covalent interaction. In someembodiments, the dye binds to a single strand of DNA or a single strandof a double-stranded DNA complex. In some embodiments, the dye binds toboth strands of a double-stranded DNA complex.

Throughout this specification, a dye bound to DNA is referred to as adye complex. In some embodiments, the dye binds to DNA with a highaffinity and a high binding constant.In some embodiments, and asmentioned herein, the affinity and concentration of the dye is greatenough that the dye is sufficient to “stain” (post penetration andDNA-binding) at least 250,000 cells/μL.

In some embodiments, the dye is fluorescent. Thus, the dye absorbsincident light at one wavelength or one group of wavelengths around apeak absorption wavelength (also referred to as a peak excitationwavelength), and emits light at another wavelength or at a group ofwavelengths around a peak emission wavelength. In some embodiments, thepeak absorption wavelength is different from the peak emissionwavelength. Furthermore, in some embodiments, the peak adsorptionwavelength and/or the peak emission wavelength is/are dependent upon theenvironment of the dye. For example, in some embodiments, the peakabsorption wavelength and/or peak emission wavelength for a dye bound toDNA (i.e., a dye complex) differs from the peak adsorption wavelengthand/or peak emission wavelength for an unbound dye.

In some embodiments, the peak adsorption wavelength and the peakemission wavelength for a dye complex differ by at least 10 nm, or atleast 15 nm, or at least 20 nm, or at least 25 nm, or at least 30 nm, orat least 35 nm, or at least 40 nm, or at least 45 nm, or at least 50 nm.In some embodiments, the peak absorption wavelength for a dye complex isin the range of 400-700 nm. For example, in some embodiments the peakabsorption is in the range of 425-550 nm, or in the range of 450-525 nm,or in the range of 475-500 nm, or in the range of 480-495 nm, or in therange of 485-490 nm. Also for example, in some embodiments the peakabsorption is in the range of 500-700 nm, or in the range of 550-675 nm,or in the range of 600-650 nm, or in the range of 620-640 nm.

In some embodiments, the peak emission wavelength for a dye complex isin the range of 425-800 nm, such as in the range of 425-700 nm. Forexample, in some embodiments, the peak emission is in the range of450-650 nm, or in the range of 475-625 nm, or in the range of 500-550nm, or in the range of 510-540 nm, or in the range of 520-530 nm.

For example, in some embodiments the fluorescent dye is selected fromthiazole orange or1-methyl-4-[(3-methyl-2-(3H)-benzothiazolylidene)methyl]quinoliniump-tosylate, thiazole blue,4-[(3-methyl-2-(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonium)propyl]quinoliniumdiiodide, 3,3′-dimethyloxacarbocyanine iodide or3-methyl-2-[3(3-methyl-2(3H)-benzothiazolylidene-1-propenyl]benzoxazolium iodide, thioflavine T, the stains SYTO® and TOTO® (LifeTechnologies), ethidium bromide, propidium iodide, acridine orange,coriphosphine O, auramine O, the stains HOECHST 33258(2′-(4-hydroxyphenyl)-5-(4methyl-1-piperizinyl)-2,5′-bi-1H-benzimidazole trihydrochloride hydrate)and HOECHST 33342®(2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperizinyl)-2,5′-bi-1H-benzimidazoletrihydrochloride), 4′6-diamino-2-phenylindole dihydrochloride (DAPI),4′,6-(diimidazolin-2-yl)-2-phenylindole dihydrochloride (DIPI),7-aminoactinomycin D, actinomycin D, and LDS 751(2-(4-(4-dimethylaminophenyl)-1,3-butadienyl)-3-ethylbenzoth iazoliumperchiorate), or a dye sold under the tradename SYBR® (LifeTechnologies, e.g., SYBR Green, SYBR Gold, SYBR 11, etc.), or the like.

In some embodiments, in addition to a dye as described above, a labelingcomposition of interest further comprises one or more of the componentsidentified in the following paragraphs. It will be appreciated that thelists below are representative only, and that substitutions may be madewhen desired using chemically and/or biologically equivalent materials.

In some embodiments, the labeling composition contains a surfactant ordetergent. Ionic and neutral surfactants can be employed. For example,zwitterionic surfactants such as steroidal glucosides (e.g., Big CHAP,Big Doxy CHAP, etc.) and glucopyranosides, as well as other surfactantsknown in the art may be used. Surfactants can be present in the labelingcomposition in any suitable amount, such as 0.0001-0.1% (w/v).

In some embodiments, the labeling composition further contains a bufferor pH modifier. Examples of buffers or pH modifiers include, but are notlimited to, PBS, MOPS, and HEPES. Additional examples of buffers or pHmodifiers include: acids such as hydrochloric acid, hydrobromic acid,acetic acid, phosphoric acid, and the like; bases such as sodiumhydroxide, sodium carbonate, sodium bicarbonate, organic amines (e.g.,imidazole, triethylamine, etc.), and the like; and salts such as sodiumchloride, calcium chloride, and the like. For example, an organic buffermaterial such as imidazole may be present in the labeling composition ata concentration in the range of about 0.001-3% (w/v). Also for example,an inorganic material such as HCl may be present in a concentration inthe range of about 0.001-5% (w/v). Similarly, a salt such as NaCl may bepresent at a concentration in the range of about 0.001-3% (w/v). Asmentioned previously, equivalents of such materials are known and may besubstituted where appropriate.

In some embodiments, the methods and materials of interest furtherinvolve additional components as necessary or desired. Examples of suchadditional components include colorants, preservatives, anti-microbialagents, osmolality adjustors, and co-solvents.

For example, an anti-microbial agent such as Proclin (e.g., Proclin 150,200, 300, etc.), vancomycin, penicillin, and the like may be present.The anti-microbial agent may be present at a concentration in the rangeof about 0.001-0.5% (w/v).

In some embodiments, stock solutions of various components may be madeduring preparation of the labeling compositions of interest. Forexample, stock solutions of the any component (e.g., dye compound, etc.)can be made, such as to aid in solubilizing the components or toaccurately measure the component volumes. Stock solutions can be madeusing water as solvent, or using an organic solvent such as dimethylsulfoxide, isopropanol, ethanol, or methanol, or using a combinationthereof.

In some embodiments, the methods and materials of interest involve anaqueous solution of the above-identified components. It will beappreciated that the concentration of the various components present inthe labeling composition can vary according to the intended use. Theparagraphs above and below provide some guidelines for concentrations ofmaterials present in the labeling composition. As indicated herein, insome embodiments the labeling composition is combined with a fluid to beanalyzed (e.g., a body fluid). Accordingly, although the guidelinesprovided herein refer to amounts of various components as present in thelabeling composition, it will be appreciated that such guidelines arealso applicable to the combination of the labeling composition and thefluid to be analyzed (i.e., in some embodiments, the material that isactually analyzed in a hematology analyzer as described herein willcontain similar percentages and relative amounts of the variouscomponents).

In some embodiments, the dye is present in the labeling composition in apredetermined concentration. It will be appreciated that thepredetermined concentration may vary depending on the identity of thedye (i.e., the chemical structure, binding affinity for the nucleic acidtarget, etc.) as well as the operating parameters of the analyzer to beused. In some embodiments, the dye component is present in an amountnecessary to stain at least the number of cells present in the bodyfluid, such as 10 cells/μL or greater, or 50 cells/μL or greater. Forexample, the dye is present in an amount necessary to stain 100,000cells/μL or greater, or 200,000 cells/μL or greater, or 250,000 cells/μLor greater. In some embodiments, the amount of dye compound present isin the range of 0.000001-0.5%, or 0.00001-0.5%, or 0.0001-0.5%, or0.001-0.1% (w/v).

In some embodiments, an organic buffering agent is present in thelabeling composition. Examples of such a material include organicamines, such as triethylamine, trimethylamine, and cyclic amines such asimidazole, and the like. The organic buffering agent (along with anyother buffers) may be present in an amount necessary to create andmaintain a desired pH in the analyte composition.

In some embodiments, the labeling composition containing a dye ofinterest is isotonic or substantially isotonic (i.e., within about 20%of isotonic, or within about 10% of isotonic, or within about 5% ofisotonic). In some embodiments, the labeling composition containing adye of interest has neutral pH or substantially neutral pH (i.e., withina pH range of about 6-8, or about 6.5-7.5). These characteristics of thelabeling composition aid in reducing damage to cells of any type duringsample preparation and analysis, and therefore result in more accuratecellular analysis of body fluid samples.

Methods

In some embodiments, the methods of interest involve contacting thepopulation of cells with the labeling composition. In some suchembodiments, the contacting involves mixing the labeling compositionwith a fluid (e.g., a body fluid) containing the cells to be analyzed.Thus, in some embodiments the methods of interest involve diluting afluid sample containing cells with a labeling composition, wherein thelabeling composition comprises water and a dye, and optionally comprisesother components such as those provided herein. In some suchembodiments, the methods involve a single dilution step (i.e. adding thelabeling composition to the fluid sample only once) at a predetermineddilution ratio that provides an optimal concentration of analyte forfluorescence detection and measurement.

In some embodiments, because the dyes of interest bind to DNA, cellsthat lack DNA are not able to form a dye complex when exposed to thedye. Cells lacking DNA that have been exposed to the dye therefore donot exhibit the emission characteristics of the dye complex whenanalyzed using the methods described herein (e.g., when exposed to afluorescence excitation energy source) or only emit minimal or weakfluorescence due to autofluorescence. In contrast, cells that containDNA are able to form a dye complex. Such cells, when stained with a dyeof interest, exhibit the emission characteristics of the dye complexwhen analyzed using the methods described herein. In view of thisdistinction, in some embodiments the methods of interest provide a meansfor differentiating cells containing DNA from cells that lack DNA. Insome embodiments, the methods of interest also provide a means fordifferentiating cells containing a nucleus from cells lacking a nucleus,particularly when the nucleus contains DNA. In some embodiments, thedifferentiation is provided based on fluorescence measurements thateither observe fluorescence from a cell (indicating the presence of DNAand possibly indicating the presence of a nucleus) or do not observefluorescence from a cell (indicating the lack of DNA and possiblyindicating the lack of a nucleus in the cell). Also, non-cellular events(e.g., non-cellular objects that lack DNA) present in the fluid beinganalyzed are differentiated by the lack of characteristic fluorescenceemission.

For example, when RBCs are exposed to the dye, mature RBCs (which lack anucleus and lack DNA) do not form the dye complex. Therefore, RBCsexposed to the dye do not exhibit the emission characteristics of thedye complex when analyzed using the methods described herein. Incontrast, WBCs contain a nucleus and DNA, and therefore form the dyecomplex when exposed to the dye. In some embodiments, therefore, themethods of interest allow differentiation of WBCs from RBCs in a samplecontaining such cells. The differentiation is based on the presence orabsence of a fluorescent signal indicative of the formation of the dyecomplex.

In addition to differentiation as described above, the formation of thedye complex between a dye of interest and DNA also allows forenumeration of cells containing DNA in a fluid sample containing cells.For example, the methods of interest include enumerating WBCs in a fluidsample containing such cells. Enumeration is based on observation offluorescence indicative of the formation of the dye complex in a methodsuitable for counting cells. Suitable methods include automatedhematology analyzers such as flow cytometers and otherfluorescence-based cell counting methods.

As mentioned herein, the methods of interest allow for enumeration anddifferentiation of WBCs in a sample containing WBCs and other objects(e.g., RBCs and non-cellular events). In some embodiments, the sample isa fluid such as a body fluid, and contains a very low concentration ofcells as described in more detail herein. In some embodiments, themethods of the present disclosure further allow for classification ofWBCs into 2-part, 3-part or 5-part differentials using multi-anglescattering technologies. In certain embodiments, RBCs can be furtherseparated from other non-RBC particles using multi-angle scatteringtechnologies.

In some embodiments, the methods of interest do not involve a lysingagent and do not involve lysing cells prior to recording data. Forexample, the methods of interest do not involve adding a lysing agent tothe fluid sample containing cells prior to analysis of the fluid sample.Thus, in some embodiments, the labeling composition does not contain alysing agent. The fluid samples that are analyzed are not lysates and donot contain cellular contents that have been released from a cell vialysis. In some embodiments, the fluid samples to be analyzed containintact RBCs and such RBCs are not lysed prior to fluorescencemeasurements. By “lysing agent” is meant to include any materials thatcause significant cell lysis, particularly (but not limited to) RBClysis. Lysing agents include, but are not limited to, various types ofsurfactants, enzymes, antibodies, pH adjusting agents, osmolytes(osmolality adjusting agents), and the like that are known in the art orlater discovered.

Because lysing agents are not involved in the preparation of fluidsamples to be analyzed, WBCs present in the fluid are not damaged asthey would or might be were lysing agents employed. This is particularlyadvantageous in body fluids containing WBCs, because the WBCs containedtherein are typically more fragile than WBCs in whole blood.

As mentioned above, in some embodiments the methods of interest involvedilution with a predetermined amount of labeling composition to providea desired dilution ratio and a desired analyte concentration. Thepredetermined amount may be determined based on detection limits in theapparatus used for analysis of the fluid sample. In some embodiments,the detection limit is optimized by adjustments to the sample injectionrate (relative to the flow cell rate), measurement duration, and thelike. For example, a prototype analyzer with 1:10 (blood:reagent)dilution ratio, 4.0 μL per second injection rate, and 60 seconds samplemeasurement (data collection), would result in a collection ofapproximately 240 events for a sample at 10 cells per μL, sufficient forsupporting precise cellular analysis.

In some embodiments, the methods of interest are suitable for analyzingany fluid sample with WBCs. As mentioned previously, such fluid samplesinclude body fluids, and also include other fluid samples such asprepared aqueous solutions of cells, plant-based and plant-derivedsolutions, and like, particularly where such sample fluids contain lowcell counts (e.g., less than 40 cells/μL, or less than 30 cells/μL,etc.).

In some embodiments, the methods of interest provide a simple, reliable,and accurate means for enumerating and differentiating WBCs in a samplefluid containing WBCs and optionally containing RBCs. Fluorescencedetection using automated hematology analyzers ensures accuracy andreliability, and the use of single-step dilution provides simplicity insample preparation. The sample fluid containing cells is stained with afluorescent label contained in a labeling composition, wherein thefluorescent label permeates cell walls and binds to DNA to create a dyecomplex. Stained WBCs emit fluorescent emission signals indicative ofthe dye complex, whereas RBCs do not emit such signals or emit suchsignals at a significantly lower intensity. The labeling composition isfree of lysing agents and no lysing agents are added to the samplefluid, thereby protecting the WBCs present. Optimal detecting limits aredetermined by adjustment of variables including sample flow rate,detection frequency, and the like.

Additional aspects of methods for analyzing WBCs and systems forperforming such methods can be found in co-pending U.S. ProvisionalPatent Application Ser. No. 61/482,541, filed May 4, 2011, as well asco-pending U.S. Provisional Patent Application Ser. No. 61/482,549,filed May 4, 2011, the disclosures of which are incorporated byreference herein in their entireties.

Output and Advantages

In some embodiments, analysis of the dyed fluids results in a variety ofinformation suitable for multi-dimensional data analysis, graphs, andthe like. For example, in some embodiments, the methods described hereinprovide an accurate total WBC count. In some embodiments, the methodsprovide data as to the relative proportions of different types of WBCsin a sample (e.g., cell counts of neutrophils, eosinophils, basophils,lymphocytes, and/or monocytes, or various combinations thereof). Suchdata may be used, for example, as diagnostic information in treatingpatients.

As mentioned herein, in some embodiments the methods of interest involveselectively staining WBCs in a fluid sample by introducing a dye thatselectively binds to DNA and fluoresces when so bound. Accordingly,analysis of a stained sample fluid includes recording the strongerfluorescence emissions that originate from cells containing a dyecomplex (e.g., WBCs) compared with the weak or non-existent fluorescenceemissions that originate from cells lacking the dye complex (e.g.,RBCs). The fluorescence measurements can be obtained using, for example,multi-channel optical scattering analyzers.

In some embodiments, the materials and methods of interest allow forcellular analysis of body fluids and other fluids useful, for example,in medical diagnostics. Cellular analysis of fluid samples with very lowcell counts (e.g., less than 40 per μL, or less than 10 per μL, etc.) isenabled by the methods herein, including body fluids having particularlyfragile cells. The simplicity of the methods allows such samples to beanalyzed within 1-2 hours after sample draw from a patient, which alsoaids in analysis of fragile cells. The lack of cell lysing agents in thecompositions and methods described herein further adds to the ability toanalyze samples containing fragile cells. Damage to fragile cells in thefluids of interest potentially results in inaccurate analysis such asinaccurate cell counts and cellular differentiation.

The optimal dilution ratios offered by the methods described herein area further improvement over traditional hematological analysis. Forexample, the dilution ratios (i.e. blood:diluent=1:300 for RBC dilution)used by traditional hematology analyzers are not sensitive enough tosupport measuring samples with very low cellular concentrations. Incontrast, in some embodiments, the methods herein allow a singledilution to provide an optimal dilution ratio for optimal fluorescencedetection.

The methods of the present disclosure are simple, e.g., in someembodiments, only a single reagent and a single dilution are required.In some embodiments, the dilution ratios can be easily adjusted and/oroptimized. Furthermore, the methods of the present disclosure are simpleto implement, and can be performed, e.g., on a traditional hematologyanalyzer, without the need for extensive adjustments and/ormodifications.

These and other advantages will be apparent to one of skill in the artbased on the disclosure provided herein, including the examples below.

EXAMPLES

General Testing Procedure: (A) Modify flow script and algorithm of aCELL-DYN Sapphire hematology analyzer to allow it to be used for a bodyfluid assay. (B) All body fluid (BF) assays are conducted using(slightly modified) WBC extended counting mode: BF sample aspiration(˜120 μL), followed by sample (BF) to reagent (Sapphire reticulocytereagent) mixing, ratio at 1 to 35, followed by mixed sample incubation(40° C. ×25 sec), followed by incubated sample delivered to flow cellfor measurement (32 sec measurement), followed by data collection, rawdata recorded as .fcs files, followed by use of software such as FCSExpress to analyze the raw data.

All reference results were measured using standard manual chambercounting procedures.

Reagent was formulated using the following components andconcentrations:

Component Concentration¹ Imidazole 0.3400% 1N HCl 2.5325% NaCl 0.6800%Proclin 300 0.0315% BIGCHAP 0.0050% SYBR 11 0.0002% DMSO² 0.0220% WaterTo 100% ¹1% = 1 g/100 mL ²DMSO (dimethyl sulfoxide) was used to make astock solution of the dye component.

Example 1 Analysis of Low Cell Count Samples

A prototype analyzer with 1:35 (blood:reagent) dilution ratio, 2.3 μLper second injection rate, and 32 seconds sample measurement (datacollection), results in a collection of more than 20 events for a sampleat 10 cells per μL.

FIGS. 2A and 2B show scattergrams of cellular analysis of a body fluidsample. The cells were enumerated after analysis: WBC=89/μL(reference=81/μL); RBC=2012/μL (reference=1733/μL). FIGS. 3A and 3B showscattergrams of cellular analysis of another body fluid sample. Thecells were enumerated after analysis: WBC=0.95/μL (reference=0.33/μL);RBC=142/μL (reference=122/μL).

Example 2 Analysis of Very Low Cell Count Samples

The detection limit was further validated in a study using diluted buffycoat samples. Six levels of diluted buffy coat samples were preparedusing serial dilutions. FL1 vs. IAS scattergrams for the six levels ofdiluted buffy coat samples are shown in FIGS. 4A-4F. FIG. 5 shows thecorrelation of WBC (measured vs. calculated). Very good correlationswere achieved: (A) Y=1.0239 X−4.9 (R²=0.9984) for all six levels and (B)Y=1.0787 X−1.5 (R²=0.9598) for the three levels with lowest cellconcentrations.

Example 3 Cellular Analysis of Body Fluid Samples at Dilution Ratio of1:35

A comprehensive body fluid study was conducted to evaluate the methodsprovided herein. A total of 91 body fluid specimens, including CSF,plural, peritoneal, and ascites fluids, were measured and analyzed on aprototype analyzer with 1:35 (blood:reagent) dilution ratio, 2.3 μL persecond injection rate, and 32 seconds sample measurement (datacollection). Reference values of WBC and RBC were achieved by manualchamber counting.

Excellent correlations in WBC were achieved between the methodsdescribed above and the reference method (FIG. 6): (A) Y=1.1305X−9.8411, R²=0.997 (Full range, up to approximately 40,000/μL); (B)Y=1.017 X+7.1395, R²=0.9765 (<2,000/μL); (C) Y=1.0899 X+1.1253,R²=0.9663 (<200/μL); and (D) Y=1.149 X+1.1461, R²=0.8459 (<50/μL). Verygood correlations in RBC were achieved between the methods describedabove and the reference method (FIG. 7): (A) Y=0.8996 X+403.62,R²=0.9595 (Full range, up to approximately 200,000/μL); (B) Y=1.0833X−11.427, R²=0.9513 (<3,000/μL); (C) Y=0.979 X−1.0747, R²=0.8284(<200/μL); and (D) Y=0.9994 X+0.5951, R²=0.6917 (<50/μL). For RBCanalysis, the samples with many RBC ghosts were not included in thecorrelation analysis.

Example 4 Cellular Analysis of Body Fluid Samples at Dilution Ratio of1:10

Another comprehensive body fluid study was conducted to evaluate themethods provided herein. A total of 155 body fluid specimens, includingCSF, plural, peritoneal, and ascites fluids, were measured and analyzedon a prototype analyzer with 1:10 (blood:reagent) dilution ratio, 2.3 μLper second injection rate, and 32 seconds sample measurement (datacollection). Reference values of WBC and RBC were achieved by manualchamber counting.

Excellent correlations in WBC were achieved, even for the samples withlow-end WBC concentrations, between the methods described above and thereference method (FIG. 8): Y=1.1327 X+1.1937, R²=0.8195 (WBC<50/μL, 72samples). Very good correlations in WBC were achieved, even for thesamples with low-end RBC concentrations, between the methods describedabove and the reference method (FIG. 9): Y=0.8244 X+0.6128, R²=0.7465(RBC<50/μL, 38 samples). For RBC analysis, the body samples with manyRBC ghosts were not included in the correlation analysis.

That which is claimed is:
 1. A method for analyzing a body fluid containing cells, the method comprising: staining a sample of a body fluid comprising fewer than 100 cells/μL, selected from cerebrospinal fluid, peritoneal fluid, pericardial fluid, pleural fluid, and synovial fluid, with a fluorescent dye, wherein the fluorescent dye is present in an amount ranging from 0.000001 to 0.5% (w (grams)/v (100 ml)), permeates a cell membrane and binds to a nucleic acid to form a dye complex within the cell; irradiating the stained sample of the body fluid with energy from an energy source; measuring a fluorescence signal emitted by the dye complex in the stained sample of the body fluid; and differentiating cells with nuclei from cells without nuclei based only on the presence or absence of the fluorescent dye.
 2. The method of claim 1, wherein the body fluid comprises fewer than about 20 cells/μL.
 3. The method of claim 1, wherein the body fluid comprises greater than about 20 cells/μL.
 4. The method of claim 1, wherein the nucleic acid is a DNA or an RNA.
 5. The method of claim 1, wherein the energy source produces monochromatic light having a wavelength in the visible spectrum, and wherein the wavelength of the monochromatic light and the wavelength of the fluorescence signal are different.
 6. The method of claim 1, wherein unbound fluorescent dye emits less fluorescent light when irradiated with energy from the energy source compared with the dye complex.
 7. The method of claim 1, wherein unbound fluorescent dye does not fluoresce when irradiated with energy from the energy source while unbound to the nucleic acid, such that cells lacking the dye complex do not emit a fluorescent signal.
 8. The method of claim 1, wherein the differentiating involves enumerating and differentiating red blood cells (RBCs) and white blood cells (WBCs).
 9. The method of claim 1, wherein the method does not involve lysing RBCs prior to the measuring.
 10. The method of claim 1, wherein the body fluid comprises intact WBCs and RBCs.
 11. The method of claim 1, wherein the measuring and differentiating is carried out using an automated hematology analyzer, a flow cytometer, or another diagnostic analyzer for body fluid samples.
 12. The method of claim 1, wherein the measuring comprises flowing the stained sample of the body fluid through a flow cell in a cytometer.
 13. The method of claim 1, wherein the fluorescent dye is provided in a composition that further comprises water.
 14. The method of claim 1, wherein the cells in the body fluid are selected from white blood cells (WBCs) and red blood cells (RBCs).
 15. The method of claim 1, wherein the method further comprises aspirating the body fluid to obtain a volume of the body fluid.
 16. The method of claim 15, wherein the volume of the body fluid is 120 μL.
 17. A method for analyzing a fluid, the method comprising: contacting a sample of a fluid comprising less than about 40 cells/μL selected from cerebrospinal fluid, peritoneal fluid, pericardial fluid, pleural fluid, and synovial fluid, with a fluorescent dye, wherein the fluorescent dye is present in an amount ranging from 0.000001 to 0.5% (w (grams)/v (100 ml)), permeates a cell membrane and binds to a nucleic acid to form a dye complex within the cell; irradiating the sample of the fluid with energy from an energy source; measuring a fluorescence signal emitted by the dye complex in the sample of the fluid; and differentiating cells with nuclei from cells without nuclei based only on the presence or absence of the fluorescent dye.
 18. The method of claim 17, wherein the fluid contains fewer than about 20 cells/μL.
 19. The method of claim 17, wherein the fluid contains fewer than about 5 cells/μL.
 20. The method of claim 17, wherein the cells in the fluid are selected from white blood cells (WBCs) and red blood cells (RBCs).
 21. A method for differentiating cells in a sample, the method comprising: contacting a sample of a fluid comprising fewer than 100 cells/μL selected from cerebrospinal fluid, peritoneal fluid, pericardial fluid, pleural fluid, and synovial fluid, with a solution comprising a fluorescent dye, wherein the fluorescent dye is present in an amount ranging from 0.000001 to 0.5% (w (grams)/v (100 ml)), is water soluble, permeates a cell membrane, and binds to a nucleic acid; irradiating the cells with an excitation light from an excitation light source; measuring light emissions from the cells; differentiating the cells that contain nucleic acids from the cells that lack nucleic acids based only on the measured light emissions; and classifying the cells as white blood cells (WBCs) or red blood cells (RBCs) based on a plurality of optical data obtained from the sample using one or more light scattering detectors.
 22. The method of claim 21, wherein the cells in the sample are selected from white blood cells (WBCs) and red blood cells (RBCs). 